A Bright Idea: Using GFP to Teach STEM€¦ · 2 VISIT for complete experiment details & free student protocols. A Bright Idea: Using GFP to Teach STEM EDVOTEK® WORKSHOP Introduction

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EDVOTEK® WORKSHOPA Bright Idea: Using GFP to Teach STEM

Introduction

THE GREEN FLUORESCENT PROTEIN

Green Fluorescent Protein, or GFP, is a small protein that possesses the ability to absorb blue light and emit green light in response. This activity, known as fl uores-cence, does not require any additional special sub-strates, gene products or cofactors to produce visible light.

GFP was fi rst isolated from the jellyfi sh Aequorea victoria in the 1970’s. Once scientists identifi ed its DNA sequence, they were able to use genetic engineering to introduce fl uorescent proteins into other organisms, such as E. coli and C. elegans. Scientists also identifi ed particular amino acid substitutions in GFP that altered the behavior of its ‘chromophore’, a special structure within the protein that is responsible for light production (Figure 1). Different changes bring about dif-ferent patterns of light absorption and emission, allowing scientists to develop a rainbow of fl uorescent proteins. For their discovery and development of GFP and other fl uorescent proteins, Osamu Shimomura, Martin Chalfi e and Roger Tsien were awarded the Nobel Prize in Chemistry in 2008.

Fluorescent proteins have become an essential tool in cell and molecular biology when coupled with fl uo-rescent microscopy studies. By tagging proteins with GFP in vivo, researchers can determine where those proteins are normally found in the cell. Similarly, using GFP as a reporter, scientists can observe biological processes as they occur within living cells. In the model organism zebrafi sh (Danio rerio), scientists use GFP to fl uorescently label blood vessel proteins so they can track blood vessel growth patterns and networks. In these ways, GFP and fl uorescent microscopy have enhanced our understanding of many biological processes by allowing scientists to watch biological processes in real-time.

USING FLUORESCENT PROTEINS FOR BIOPROCESSING

Scientists utilize genetic engineering and recombinant DNA techniques to effectively re-program cells to create living factories, capable of producing large amounts of important proteins like biopharmaceuticals. The demand for protein products has driven development of novel technologies for microbial growth, large-scale production of biomolecules, and the subsequent purifi cation of those molecules.

In biotechnology laboratories, fl uorescent “tags” simplify purifi cation of important proteins. Using mo-lecular cloning techniques, the gene of interest is isolated and “tagged” with fl uorescent proteins at their N- or C- terminus. The recombinant DNA is introduced into cells, where it serves as a template for protein production. After several hours, the cell’s walls and membranes are disrupted to release its intracellular

Background Information

Bring exciting STEM learning techniques into your classroom laboratory! In this hands-on workshop, we will build a size-exclusion chromatography column. The column is used to purify green fl uorescent protein (GFP) from a crude bacterial extract. Proteins containing fractions are identifi ed by fl uorescence and analyzed for purity by SDS-PAGE.

Figure 1: GFP Chromophore

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EDVOTEK® WORKSHOP A Bright Idea: Using GFP to Teach STEM


components. The resulting “lysate” consists of cytoplasm, metabolites, DNA, RNA, proteins and organelles. GFP-tagged proteins can be isolated from the lysate by passing it over chromatography columns. The pro-tein-containing fractions are easy to identify because the GFP-labeled protein can be tracked using UV light.

In this experiment, Size Exclusion Chromatography (or SEC) is used to purify GFP from a crude bacterial cell lysate. SEC is a biochemical technique used to separate proteins into fractions by size. To perform SEC, a column is packed with tiny, porous beads. The crude cell lysate is placed onto the top of the column and fl owed through the column. Large proteins freely fl ow around the beads and through the column, where they can be collected in early fractions that are eluted from the column. In contrast, the pores in the beads periodically retain small proteins. This slows their progress through the column, and the smaller proteins elute from the column in later fractions (Figure 2).

In most SEC experiments, the protein content of a fraction is determined by measuring the absorbance of the fractions at 280 nm. However, in the case of GFP-tagged proteins, we can monitor elution using a hand held long wave UV light. The fl uorescent proteins will be detected on the column and subsequently in the test tubes by examination under long UV light.

After the elution is complete, SDS-Polyacrylamide Gel Electrophoresis (or SDS-PAGE) is used to confi rm the presence of GFP in the collected fractions (Figure 3). Proteins produce a unique challenge for electrophoresis because they have complex shapes and different charges,

Figure 2: Different-sized molecules moving through the column.

Small molecules

Mediummolecules

Largemolecules

Column

Elution Buffer

Porous Beads(Matrix)

Figure 3: An overview of SDS-PAGE.

Stain to visualize..Protein Bands are separated by size..

Protein

Add SDS

Then, load samples onto SDS-PAGE gel.

Negatively charged SDS molecules



which affect how they migrate through the gel. In order to accurately separate proteins by molecular weight and not by shape or charge, the secondary structure of the protein is unfolded using the anionic detergent sodium dodecyl sulfate (SDS) and a reducing agent. The SDS molecules form a complex with the protein, negating its inherent charge. The reducing agent breaks covalent bonds that link protein sub-units.

After denaturation, the mixture of proteins is added into depressions (or “wells”) within a gel, and then an electrical current is passed through the gel. Because the SDS-protein complex has a strong negative charge, the current drives the proteins through the gel towards the positive electrode. At fi rst glance, a polyacryl-amide gel appears to be a solid. On the molecular level, the gel contains channels through which the proteins can pass. Small proteins move through these holes easily, but large proteins have a more diffi cult time squeezing through the tunnels. Because molecules of different sizes travel at different speeds, they separate into discrete “bands” within the gel. After the current is stopped, the bands are visualized using a stain that sticks to proteins.

In this workshop, we will use GFP and SEC to bring exciting STEM learning techniques into your classroom laboratory! First, we will build a size-exclusion chromatography column. The column is used to purify GFP from a crude bacterial extract. Protein containing fractions are identifi ed by fl uorescence and analyzed for purity by SDS-PAGE.


STEM HIGHLIGHTS

SCIENCE• Transformation of Recombinant

DNA into Bacteria• Gene Expression using Inducible

Promoters

TECHNOLOGY• Size Exclusion Chromatography• Innovations in Biotechnology (Green Fluorescent Protein)• Separation of Proteins by Size using SDS-PAGE• Pipetting

ENGINEERING• Biochemical Engineering• Genetic Engineering • Separation Processes

MATHEMATICS• Making Measurements• Plotting Data• Molecular Weight Determination using a Standard Curve



Packing a Chromatography Column

The loading of the column and subsequent elution will be done at room temperature. The elution buffers and the fractions collected will be stored on ice as they elute from the column.

1. Vertically mount the column. Make sure it is straight.

2. Slide the cap onto the spout at the bottom of the column. Fill about one-third of the column with the elution buffer.

3. Mix the slurry thoroughly by swirling or gently stirring.

4. Carefully pipet the mixed slurry into the column by letting it stream down the inside walls of the col-umn. If the fl ow is stopped by an air pocket, stop adding the slurry and fi rmly tap the column until the air is removed and the slurry continues to fl ow down the side of the column.

5. Place an empty beaker under the column to collect wash buffer.

6. Remove the cap from the bottom of the column and allow the matrix to pack into the column.

7. Wash the packed column with 5 ml of 1x elution buffer. Do not allow the column to dry.

8. When a small amount of buffer remains above the resin, slide the cap onto the spout to prevent drips.

NOTE:The loading of the column and subsequent elution will be performed at room temperature. The fractions will be stored on ice as they are eluted from the column.



Collecting Column Fractions

1. Label the set of eight microcentrifuge tubes 1 through 8.

2. Slowly load the column with 0.2 ml of the protein extract. Remove the cap from the column and allow the extract to completely enter the column.

3. Elute the proteins using 1x elution buffer. Add buffer slowly (several drops at a time) to avoid dilut-ing the protein sample. Be sure to continually add buffer to the column to elute the proteins. Do not allow the column to dry. A hand-held UV light can monitor the progress of the fl uorescent protein as it travels through the column.

4. Using the graduated marks on the sides of the tubes, collect 0.5 ml fractions in the labeled microcentri-fuge tubes.

5. Store fractions on ice immediately upon collection.

6. Check all fractions using long wave UV light to identify tubes that contain the fl uorescent protein.

7. Save the fractions containing the purifi ed protein for further analysis by SDS-PAGE.

Sample Preparation for SDS-Polyacrylamide Gel Electrophoresis1. Using a UV light, identify the tube with the highest level of green fl uorescence.

2. Transfer 200 µl each of the eluate into two clean microcentrifuge tubes. Label the fi rst tube “gfp na-tive” and the second tube “gfp denatured”.

3. Add 25 µl of 50% glycerol to the tube labeled “gfp native”. Mix well and set on ice.

4. Add 25 µl of protein denaturing solution to the tube labeled “gfp denatured”. The denaturing solu-tion contains detergents and reducing agents, which break down the protein secondary and tertiary structure.



NOTE: DO NOT BOIL “GFP NATIVE” SAMPLES!

1. Using a hot plate or microwave, HEAT a beaker of water until it boils. 2. COVER with aluminum foil and carefully remove from heat.3. Tightly CAP “gfp denatured” sample tubes. PUSH tubes through foil to suspend

in the boiling water. DO NOT BOIL “GFP NATIVE” SAMPLES!4. INCUBATE the samples for 5 minutes. 5. Immediately PROCEED to loading the gel while the samples are still warm.

Heat1. 2. Cover

with foilProceedto Gel

Loading

3. 4. 5.

5min.

Wear gloves and safety goggles

Protein Denaturation

Purifi cation & Size Determination of Green & Blue Fluorescent ProteinsFor 6 Groups. When bacteria are used to make medicinally useful proteins by transformation, the protein of interest must be separated from all of the other cellular proteins. In this experiment, the unique fl uorescent properties of Green Fluorescent Protein (GFP) and Blue Fluorescent Protein (BFP) will be used as an assay during their purifi cation from the extract of genetically modifi ed strain of E.coli. The column fractions containing GFP or BFP will be identifi ed by fl uorescence and then purifi ed. As an additional activity, purifi ed protein fractions can be sepa-rated by SDS polyacrylamide gel electrophoresis (SDS-PAGE) to estimate the purity and size of the GFP and BFP proteins.

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1. Using a fresh pipet tip, MEASURE 20 µl of the Standard Protein Marker (A).2. PLACE the pipet tip under the buffer and directly above the sample well, resting gently against the back

plate of the gel cassette.3. Slowly DISPENSE the sample by depressing the plunger.4. REPEAT steps 1-3 with remaining samples, changing the tip between each new sample.5. Once all samples have been loaded, carefully PLACE the cover onto the electrode terminals. 6. CONNECT the electrical leads to the power supply. 7. SET the voltage of the power supply and PERFORM elec-

trophoresis (See Table A for time and voltage guidelines). Allow the proteins to separate on the gel for the recom-mended length of time, or until the tracking dye reaches the bottom of the gel.

8. TURN OFF the power supply and carefully REMOVE the lid. The gel can now be removed from the chamber and ana-lyzed.

For easier analysis the gel can be removed from the cassette. Using a metal spatula or screwdriver carefully pry apart the two plastic plates and gently remove the gel for analysis. The gels are very fragile and must be handled with care.

NOTE: Before staining with Protein InstaStain, shine a UV light on your gel. The GFP in its native form should fl uoresce under UV light, whereas the denatured proteins will not.

6. 7. 8.

Lane

1

2

3

4

5

Sample 1 Standard Protein Marker

Sample 2 gfp native

Sample 3 gfp denatured

Sample 4 gfp native

Sample 5 gfp denatured

Table 2: Gel Loading

Time and Voltage Guidelines

MinimumVolts

100 125 150

80 min.60 min. 50 min.

Table

ARecommended Time

Optimal

95 min. 75 min. 60 min.

Loading the Protein Samples

2 0

.0

1. 2.

2 0.0

3. 4. Repeatsteps 1-3

with remainingsamples.

5.5.



1. Place gel into a small tray with 100 ml fi xative solution. Gently fl oat a card of Protein InstaStain® into the liquid, stain side down. Remove the card after 30 sec.

2. Gently agitate on a rocking platform 1-3 hours or over-night. (Cover the tray with plastic wrap to prevent evapo-ration.)

3. After staining, protein bands will appear medium to dark against a light background.

1. 2. 3.

Staining with Protein InstaStain®

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Includes:2 Cat. # 581 MV10 Protein Electrophoresis Apparatus 1 Cat. # 509 DuoSource™ 150 (75/150 V for 1 or 2 units)4 Cat. # 590 Variable Micropipets (5 - 50 µl)

Supports up to 16 Students!



The native GFP sample runs at approximately 40 kD and the denatured GFP sample runs at approximately 27 kD.

First Student Group

Lane 1 20 µl of Standard Protein Markers (boiled for 5 min.)Lane 2 20 µl of gfp native (not boiled)Lane 3 20 µl of gfp denatured (boiled for 5 min.)Lane 4 20 µl of gfp native (not boiled)Lane 5 20 µl of gfp denatured (boiled for 5 min.)

Second Student Group

Lane 6 20 µl of Standard Protein Markers (boiled for 5 min.)Lane 7 20 µl of gfp native (not boiled)Lane 8 20 µl of gfp denatured (boiled for 5 min.)Lane 9 20 µl of gfp native (not boiled)Lane 10 20 µl of gfp denatured (boiled for 5 min.)

When protein samples are boiled for 5 minutes in the presence of SDS and 2-mercaptoethanol, proteins lose their tertiary structure and are denatured. In the absence of boiling, complete denatur-ation is not achieved and local native structures can be maintained. The fl uorescence observed in lanes 2, 4, 7 and 9, where samples were not boiled, could be due to core native structures that are responsible for the green fl uorescence of the gfp protein.

Experiment Results



Determination of Protein Molecular WeightFor 6 Groups sharing 3 gels. Using prestained LyphoProteins™, subunit molecular weights are determined by analysis using denaturing SDS vertical polyacrylamide gel electrophoresis. Prestained Proteins with unknown molecular weights are as-signed molecular weights based on the relative mobility of prestained standard protein markers.


Related Products

Construction and Cloning of a DNA RecombinantFor 5 Plasmid Constructs & Analyses. Cloning is frequently performed to study gene structure and function, and to enhance gene expression. This experiment is divided into fi ve modules. Clones are constructed by ligation of a vector and a fragment insert. The constructs are then transformed into competent cells and the cells are grown and selected for resistance. Plasmid DNA is then isolated from the transformants, cleaved with restriction enzymes, and analyzed by agarose gel electrophoresis. Recommended for college level courses. Requires wet ice ship-ment for next day delivery (by 3:00 pm in most areas).


Fermentation and Bioprocessing of Chromogenic ProteinsFor 5 Groups. Bioprocessing is the production and isolation of desired products from living cells. In this introduction to bioprocessing, students will use small-scale fermenters to produce chromogenic proteins using Escherichia coli. Protein extracts will then be separated using column chromatography to analyze the success of the fermentation process. Finally, the protein solutions will be examined by SDS poly-acrylamide gel electrophoresis to determine the purity of the chromogenic proteins.


Principles of Gel Filtration ChromatographyFor 10 Groups. Introduce chromatographic separation to your class and show them how dyes of different colors separate on the basis of their size and shape. This experiment contains materials for dye separation which include dye sample, elution buffer and plastic disposables. Columns may be rinsed and reused.




MV10 Vertical Protein Electrophoresis ApparatusOur newly redesigned MV10 is the most user-friendly vertical protein electrophoresis unit with a simple gel clip system. It runs one vertical polyacrylamide gel. All parts are color coded to ensure proper orientation.


DuoSource™ 150 Power Supply (75/150 V)The DuoSource™ is a great value for protein electrophoresis! This power supply can run two MV10 units at once. Choose between 75 or 150 V.


EDVOTEK® Variable MicropipetOur newly redesigned Variable Micropipet is easy to use, sturdy, highly accurate! Volume ranges from 5 - 50 µl and uses standard micropipet tips.


Precast Polyacrylamide GelsThree 12% Precast Polyacrylamide Gels. 9 x 10 cm.


Long Wave UV Mini-LightA hand-held UV light that is used to detect hydrolysis of the fl uo-rescent substrate and fl uorescent Artemia and Daphnia after their ingestion. Also useful for observing fl uorescence in Green (gfp) and Blue (bfp) fl uorescent proteins. Cat. #969 $35 www.edvotek.com/969

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A Bright Idea: Using GFP to Teach STEM€¦ · 2 VISIT for complete experiment details & free student protocols. A Bright Idea: Using GFP to Teach STEM EDVOTEK® WORKSHOP Introduction

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